Methods for detecting contamination by adventitious agents in mammalian cell culture for influenza vaccine production are described in reference 1. These contaminating viruses include mammalian Reoviridae, in particular orthoreoviruses (e.g. mammalian reoviruses—MRV), which would be able grow particularly well in the MDCK cells used for Optaflu™ vaccine production were they to become contaminated. At present, however, there is no available PCR method to detect MRV that is acceptable in terms of specificity and sensitivity for the purpose of quality control of influenza vaccine production.
Two known mammalian reovirus PCR assays [2, 3] have been tested by the inventors for suitability for the purpose of quality control of influenza vaccine production.
Reference 2 describes a nested PCR method, in which the first PCR with primers L1.rv5 and L1.rv6 amplify a conserved 416 bp product of the L1 gene. The nested PCR with primers L1.rv7 and L1.rv8 amplify a conserved 344 bp product of the L1 gene. The present inventor has found that, in the method described in reference 2, the first PCR yielded nonspecific products with some strains and the nested PCR was sufficiently sensitive and specific only for the MRV-1 strain Dearing and MRV-2 strain Lang. Furthermore, he found that there was reduced performance and nonspecific products with MRV-3 strain Jones.
Reference 3 describes a RT-PCR method to detect a region in the L3 inner capsid gene. The present inventor found that this method provides a low yield with significant contamination from primer-dimers.
The results of the analysis of the methods of references 2 and 3, and of the present invention, are shown in FIG. 1.
There therefore remains a need for a specific and sensitive generic method to detect and quantify all MRV types.